Controlled electroporation and mass transfer across cell membranes

ABSTRACT

Electroporation is performed in a controlled manner in either individual or multiple biological cells or biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general. In addition, a particular method and apparatus are disclosed in which electroporation and/or mass transfer across a cell membrane are accomplished by securing a cell across an opening in a barrier between two chambers such that the cell closes the opening. The barrier is either electrically insulating, impermeable to the solute, or both, depending on whether pore formation, diffusive transport of the solute across the membrane, or both are sought. Electroporation is achieved by applying a voltage between the two chambers, and diffusive transport is achieved either by a difference in solute concentration between the liquids surrounding the cell and the cell interior or by a differential in concentration between the two chambers themselves. Electric current and diffusive transport are restricted to a flow path that passes through the opening.

CROSS REFERENCES

This application is a continuation of our earlier filed application Ser.No. 10/407,580 filed Apr. 3, 2003, which is a continuation of ourearlier filed application Ser. No. 09/863,117 filed May 22, 2001 (issuedas U.S. Pat. No. 6,562,604 on May 13, 2003), which application is acontinuation of our earlier filed application Ser. No. 09/358,510 filedJul. 21, 1999 (issued as U.S. Pat. No. 6,300,108 on Oct. 9, 2001), whichapplications are incorporated herein by reference and to whichapplications we claim priority under 35 USC §120.

This invention resides in the fields of electroporation and masstransfer across cell membranes.

BACKGROUND OF THE INVENTION

Electroporation is a technique that is used for introducing chemicalspecies into biological cells, and is performed by exposing the cells toan electric potential that traverses the cell membrane. While itsmechanism is not fully understood, electroporation is believed toinvolve the breakdown of the cell membrane lipid bilayer leading to theformation of transient or permanent pores in the membrane that permitthe chemical species to enter the cell by diffusion. The electricpotential is typically applied in pulses, and whether the pore formationis reversible or irreversible depends on such parameters as theamplitude, length, shape and repetition rate of the pulses, in additionto the type and development stage of the cell. As a method ofintroducing chemical species into cells, electroporation offers numerousadvantages: it is simple to use; it can be used to treat wholepopulations of cells simultaneously; it can be used to introduceessentially any macromolecule into a cell; it can be used with a widevariety of primary or established cell lines and is particularlyeffective with certain cell lines; and it can be used on bothprokaryotic and eukaryotic cells without major modifications oradaptations to cell type and origin. Electroporation is currently usedon cells in suspension or in culture, as well as cells in tissues andorgans.

Electroporation is currently performed by placing one or more cells, insuspension or in tissue, between two electrodes connected to a generatorthat emits pulses of a high-voltage electric field. The pore formation,or permealization, of the membrane occurs at the cell poles, which arethe sites on the cell membranes that directly face the electrodes andthus the sites at which the transmembrane potential is highest.Unfortunately, the degree of permealization occurring in electroporationvaries with the cell type and also varies among cells in a givenpopulation. Furthermore, since the procedure is performed in largepopulations of cells whose properties vary among the individual cells inthe population, the electroporation conditions can only be selected toaddress the average qualities of the cell population; the procedure ascurrently practiced cannot be adapted to the specific characteristics ofindividual cells. Of particular concern is that under certainconditions, electroporation can induce irreversible pore formation andcell death. A high electric field, for example, may thus produce anincrease in transfection efficiency in one portion of a cell populationwhile causing cell death in another. A further problem with knownmethods of electroporation is that the efficiency of transfection byelectroporation can at times be low. In the case of DNA, for example, alarge amount of DNA is needed in the surrounding medium to achieveeffective transformation of the cell.

Many of the problems identified above are a consequence of the fact thatthe process of electroporation in both individual cells and tissuescannot be controlled in real time. There are no means at present toascertain in real time when a cell enters a state of electroporation. Asa result, the outcome of an electroporation protocol can only bedetermined through the eventual consequences of the mass transferprocess and its effect on the cell. These occur long after the masstransfer under electroporation has taken place. These and otherdeficiencies of current methods of electroporation are addressed by thepresent invention.

Also relevant to the present invention are current techniques for thestudy and control of mass transfer across cell membranes. Knowledge ofmass transfer across cell membranes in nature, both in cells that arefunctioning normally and in diseased cells, is valuable in the study ofcertain diseases. In addition, the ability to modify and control masstransfer across cell membranes is a useful tool in conducting researchand therapy in modern biotechnology and medicine. The introduction orremoval of chemical species such as DNA or proteins from the cell tocontrol the function, physiology, or behavior of the cell providesvaluable information regarding both normal and abnormal physiologicalprocesses of the cell.

The most common method of effecting and studying mass transfer across acell membrane is to place the cell in contact with a solution thatcontains the compound that is to be transported across the membrane,either with or without electroporation. This bulk transfer method doesnot permit precise control or measurement of the mass transfer acrossthe membrane. The composition of the solution at specific sites is notknown and is variable. In addition, when an electric field is present,the local field intensity will vary from one point to another.Furthermore, the surface of the cell that is exposed to the solution isnot well defined. Cell surface areas vary among cells in a givenpopulation, and this leads to significant differences among the cells inthe amount of mass transfer. For these reasons, the amount of masstransfer achieved by bulk transfer processes is not uniform among cells,and the actual amount transferred for any particular cell cannot bedetermined.

Attempts made so far to overcome the limitations of bulk transfertechniques include techniques for treating individual cells that includeeither the mechanical injection (microinjection) of chemical compoundsthrough the cell membrane or electroporation with microelectrodes. Ininjection techniques, the membrane is penetrated with a needle todeliver a chemical agent, localizing the application of the chemicalagent to a small region close to the point of injection. This requiresmanipulation of the cell with the human hand, a technique that isdifficult to perform, labor-intensive, and not readily reproducible.Electroporation with microelectrodes suffers these problems as well asthe lack of any means to detect the onset of electroporation in anindividual cell. These problems are likewise addressed by the presentinvention.

SUMMARY OF THE INVENTION

The present invention arises in part from the discovery that the onsetand extent of electroporation in a biological cell can be correlated tochanges in the electrical impedance (which term is used herein to meanthe ratio of current to voltage) of the biological cell or of aconductive medium that includes the biological cell. An increase in thecurrent-to-voltage ratio across a biological cell occurs when the cellmembrane becomes permeable due to pore formation. Likewise, a decreasein the current-to-voltage ratio through a flowing conductive fluidoccurs when the fluid draws a biological cell into the region betweenthe electrodes in a flow-through electric cell. Thus, by monitoring theimpedance of the biological cell or of an electrolyte solution in whichthe cell is suspended, one can detect the point in time in which poreformation in the cell membrane occurs, as well as the relative degree ofcell membrane permeability due to the pore formation. This informationcan then be used to establish that a given cell has in fact undergoneelectroporation, or to control the electroporation process by governingthe selection of the voltage magnitude. This discovery is also useful inthe simultaneous electroporation of multitudes of cells, since itprovides a direct indication of the actual occurrence of electroporationand an indication of the degree of electroporation averaged over themultitude. The discovery is likewise useful in the electroporation ofbiological tissue (masses of biological cells with contiguous membranes)for the same reasons.

The benefits of this process include a high level of control over theonset and degree of electroporation, together with a more detailedknowledge of the occurrence and degree of permeability created inparticular individual cells or cell masses. When applied to individualcells or to a succession of individual cells, this process assures thatthe individual cells are indeed rendered permeable and are indeedtransformed by the introduction of chemical species. The process alsooffers the ability to increase the efficiency of electroporation byavoiding variations in the electrical environment that would destroysome cells while having an insufficient effect on others.

In some of its more specific embodiments, the present invention involvesthe use of an electrical cell in which a biological cell can be placedand that contains a barrier that directs the electric current flow andhence the ion flow through a flow path that passes through thebiological cell while permitting substantially no electric current tobypass the biological cell. In some of these embodiments, the inventioninvolves the use of an apparatus containing two liquid-retainingchambers separated by a barrier that is substantially impermeable to anelectric current. The barrier contains an opening that is smaller thanthe biological cell such that the biological cell once lodged in theopening will plug or close the opening. To achieve electroporation, thebiological cell is secured over the opening by mechanical or chemicalmeans, preferably in a reversible manner so that the biological cell canlater be removed without damage to the biological cell. Once thebiological cell is secured over the opening, a voltage is imposedbetween the two chambers and across the biological cell residing in theopening. The passage of current between the chambers is thus restrictedto a path passing through the opening and hence through the biologicalcell. By monitoring the current-voltage relation in the electric cell,the onset of electroporation is detected and the degree of poreformation is controlled, to both assure that electroporation isoccurring and to prevent excessive pore formation and cell death. Theuser is thus afforded a highly precise knowledge and control of thecondition of and the flux across the biological cell membrane.

In another series of embodiments, this invention is useful in thediffusive transport of chemical species into or out of a biologicalcell. In these embodiments, the cell is again divided into two chambersseparated by a barrier, and the biological cell is lodged across anopening in the barrier in such a manner that the passage of liquidaround the cell from one chamber to the other is substantiallyprevented. A liquid solution of the species to be introduced into thebiological cell is placed in one or both of the chambers. Theconcentration of the species in the solution differs from that in thecell (either higher or lower, depending on whether one seeks tointroduce or remove the species from the cell), or the concentration inone chamber differs from that in the other chamber.

In preferred methods of applying this invention to diffusive transport,the solutions in the two chambers differ in concentration such that thedriving force for the diffusive transport is between the two chambersthemselves rather than between the chambers and the interior of thebiological cell. Knowledge and controlled monitoring of theconcentrations in each of the two chambers on a periodic or continuousbasis as the diffusion proceeds, together with the precise knowledge ofthe dimensions of the opening, enables the user to precisely observe andcontrol the rate and amount of the species that enters the cell. Thediffusion time can be controlled by imposing stepwise changes in theconcentrations in either or both of the chambers, thereby imposing orremoving the concentration differential. An application of particularinterest is the combination of this type of diffusive transport of achemical species with controlled electroporation as described in thepreceding paragraph.

Each of the various embodiments of this invention may be used with twoor more biological cells simultaneously, or cell masses such as intissue, rather than just one cell. The apparatus described above can beadapted for use with two or more biological cells by arranging thebarrier such that the current or diffusive transport will be restrictedto a flow path that passes through all of the cells while preventingbypass around the cells. A further application of the concepts of thisinvention is the electroporation of biological cells suspended in aflowing liquid. Electrodes are placed in fixed positions in the flowchannel, and a voltage is imposed between the electrodes while currentpassing between the electrodes is monitored. Biological cells enteringthe region between the electrodes will lower the current, the impedanceserving as an indication of the presence of one or more cells in theregion, and optionally also as a signal to initiate the application of ahigher voltage sufficient to achieve electroporation.

Among the advantages that this invention offers relative to the priorart are the ability to treat cells individually and to adapt thetreatment conditions to the needs of individual cells. In embodimentswhere voltage is applied, the monitoring of the impedance affords theuser knowledge of the presence or absence of pores and shows theprogress of the pore formation and whether irreversible pore formationthat might lead to cell death has occurred. An advantage of thebarrier-and-opening apparatus is its highly efficient use of electricalenergy by virtue of its restriction of the current to a current flowpath passing through the opening. A still further advantage is theability of the apparatus and method to be integrated into an automatedsystem whereby the condition of each cell is monitored byinstrumentation and individual cells are lodged in the opening and thenremoved at times governed by the monitored conditions.

These and further features, advantages and objects of the invention willbe better understood from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross section of a microdiffusion device useful in thepractice of the present invention for infusing a biological cell with achemical species without the assistance of an electrical current toeffect electroporation.

FIG. 2 is a cross section of a microelectroporation device useful in thepractice of the present invention for achieving pore formation in abiological cell, and optionally for infusing the cell with a chemicalspecies with the assistance of electroporation.

FIG. 3 a is a longitudinal cross section of an electroporation device inaccordance with this invention, designed for a mobile suspension ofbiological cells. FIG. 3 b is a transverse cross section of the deviceshown in FIG. 3 a.

FIG. 4 is a plot of current vs. voltage in a series of electroporationexperiments conducted using a microelectroporation device of thestructure similar to that of FIG. 2.

FIGS. 5 a, 5 b, 5 c, and 5 d are plots of current vs. voltage in afurther series of electroporation experiments conducted using amicroelectroporation device similar to that of FIG. 2.

DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS

While this invention extends to a variety of structures, methods, andapplications, this portion of the specification will illustrate certainspecific structures and methods in detail, from which the concepts ofthe invention as a whole will become apparent.

The first structure that will be discussed is an electroporation cellwith an internal support to hold a single biological cell and aninternal barrier that restricts the electric current flow in theelectric cell to a flow path that passes through the biological cell.When no voltage is applied, the structure can be used for diffusivetransport alone, unassisted by voltage-induced pore formation.

The configuration of the barrier, and the two chambers in embodimentsthat include two chambers, is not critical to the invention, and canvary widely while still serving the purposes and advantages of theinvention. Since biological cells are microscopic in size, however, thepreferred type of apparatus for the practice of this invention in eachof its various forms is one in which the structure as a whole and/or itschambers are the size of electronic chips, fabricated bymicrofabrication techniques such as those used in electronic chipmanufacture. It is further preferred that the chambers are constructedas flow-through chambers to allow the passage of the liquids incontinuous flow, intermittent flow, or flow at the direction of theuser, and to allow changes in the concentrations, pressure, and otherconditions as needed to achieve close control over the passage ofspecies across the biological cell membrane. Accordingly, a preferredstructure and method of manufacture of the apparatus are those thatinvolve the formation of the apparatus in layers or platelets withappropriate openings that form flow passages when the layers orplatelets are bonded together.

Flow-through chambers offer the advantage of permitting the successiveentry and removal of individual cells so that large numbers of cells canbe treated in succession. Flow-through chambers also permitreplenishment of solute-depleted solutions so that concentrationgradients can be continuously maintained when desired. A furtherfunction that can be served by flow-through chambers is the increase anddecrease of pressure, a function that is useful for various purposes asdescribed below.

The support for the biological cell in this structure can be anystructure that secures the biological cell in a fixed position and thatallows the passage of electric current. The most convenient support isan opening in the barrier. Securement of a biological cell over theopening serves to close, seal or plug the opening, thereby directing thepassage of electric current, diffusive transport, or both, through thecell and eliminating or minimizing leakage around the cell. A convenientmechanical means of achieving this is to impose a pressure differentialacross the opening in a direction that will press the cell against theopening. The diameter of the opening will be smaller than that of thecell, and the cell upon entering the apparatus will pass into one of thetwo chambers. By increasing the pressure in the chamber in which thecell resides, or lowering the pressure in the other chamber, the cellwill be forced against the opening, closing it off. Once the procedureis completed, the cell is readily released from the opening byequalizing the pressures in the two chambers or by reversing thedifferential such that the higher pressure is in the chamber other thanthe chamber in which the cell was introduced. The flow of liquid in thechamber in which the cell was introduced will then remove the cell fromthe opening, exposing the opening for another cell.

An alternative method of sealing the opening with the cell is by the useof a coating on the barrier surface, or over the rim of the opening, ofa substance that binds to the cell membrane. Since biological cellmembranes are negatively charged, the coating may be a substance thatbears a positive charge, such as polylysine, polyarginine, orpolyhistidine. The biological cell can be directed to the opening by apressure differential across the opening, and held in place by thecoating. One the procedure is completed, the cell can be released fromthe coating by momentarily increasing the flow rate of the liquid in thechamber on the cell side of the opening, or by imposing a reversepressure differential across the opening to urge the cell away from theopening.

The size of the opening is not critical to the invention provided thatthe opening exposes sufficient surface area on the cell membrane toachieve the desired degree of either mass transfer, the passage of anelectric current, or both, within a controllable and economicallyreasonable period of time. The optimal size will thus vary with theparticular cells being treated or studied. In general, the opening ispreferably circular or approximately circular in shape, and depending onthe cell size, preferably ranges in diameter from about 1 micron toabout 100 microns, more preferably from about 1 micron to about 50microns, and most preferably from about 2 microns to about 20 microns.The barrier in which the hole is formed and which separates the twochambers is preferably of a rigid dielectric material that isimpermeable to both water and solutes and that will hold a pressuredifferential sufficient to secure a cell against the opening. Fordevices that are manufactured by microfabrication techniques, aconvenient material for the barrier is silicon nitride. Other materialsthat will serve equally well will be readily apparent to those skilledin the art.

A further feature of preferred embodiments of this invention is the useof apparatus made of transparent materials. This enables the user toobserve cell interiors and the processes of microdiffusion andmicroelectroporation through a microscope as they occur.

An example of a microdiffusion apparatus in accordance with thisinvention for a single biological cell, for transporting materialsacross the cell membrane without the application of an electric field,is shown in FIG. 1. This components of this apparatus, from the bottomup, are an acrylic base 11, an intermediate silicon layer 12 (1 micronin thickness) with a portion 13 carved out to define the lateralboundaries of the lower of the two liquid chambers, a silicon nitridelayer 14 serving as the barrier between the two chambers, a siliconwasher 15 defining the lateral boundaries of the upper liquid chamber16, and a glass cover plate 17. A hole 18 in the silicon nitride barrierserves as the opening, and a cell or contiguous cell mass such as tissue19 is shown covering the hole. Channels extend through the acrylic baseto serve as inlet and outlet channels for the liquids that pass throughthe upper and lower chambers, as shown by the arrows in the Figure.

When the pressure in the upper chamber 16 is higher than that in thelower chamber 13, the cell will be retained in position over the hole,serving as a plug separating the liquids in the two chambers from eachother. When the composition of the solutions in the two chambers differsfrom that of the cell interior, mass transfer occurs across the cellmembrane between the chambers and the cell. When the composition of thesolution in one chamber differs from that in the other, mass transferoccurs through the cell from one chamber to the other. By preciselycontrolling the compositions of the solutions in the two chambers, onecan precisely control the mass transfer rate and direction within thecell. Since the diameter of the opening 18 is known, one can preciselydetermine the mass transfer that occurs through the opening.

The numerous applications of this microdiffusion device will be readilyapparent. For example, the device can be used to infuse a cell with acryopreservative such as glycerol by filling the upper chamber 16 withphysiological saline and the lower chamber 13 with glycerol. When usinga cell 19 for which the mass transfer coefficient of glycerol across thecell membrane is known, one can readily calculate the amount of glycerolthat will enter the cell and adjust the concentrations and exposuretimes to infuse the cell with the amount that is known to be requiredfor cryopreservation.

An example of a microelectroporation apparatus in accordance with thisinvention for a single biological cell, is shown in FIG. 2. Theapparatus is similar in construction to the microdiffusion apparatus ofFIG. 1. Its structural components, from the bottom up, are an acrylicbase 21, a lower silicon layer 22 with a portion carved out to definethe lateral boundaries of the lower liquid chamber 23, a silicon nitridelayer 24 (1 micron in thickness) serving as the barrier between the twochambers, an upper silicon layer 25 defining the lateral boundaries ofthe upper liquid chamber 26, and a cover consisting of an n+poly-silicon layer (5,000 Å in thickness) 27 and a silicon nitride layer(1 micron in thickness) 28. A hole 29 in the silicon nitride barrier 24serves as the opening, and a cell 30 (or cell mass) covers the hole.Channels extend through the acrylic base to serve as inlets and outletsfor the liquids that pass through the upper and lower chambers, as shownby the arrows in the Figure. A further layer of n+ poly-silicon (5,000Å) 31 resides above the acrylic base 21, and this layer, together withn+ poly-silicon layer 27 above the upper chamber 26 serve as the twoelectrodes. Each electrode is joined by electric leads to a printedcircuit board 32 which controls the voltage applied between theelectrodes and measures the current passing between them.

The microelectroporation apparatus shown in FIG. 2 can be fabricated byconventional microfabrication techniques, typically involving chemicalvapor deposition, masking, etching and sputtering. The operation of theapparatus will be analogous to the operation of the microdiffusionapparatus of FIG. 1. The movement of biological cells through theapparatus is achieved by suspending the cells in the liquid used to fillthe upper chamber, and cells are drawn to the opening, one at a time, byimposing a pressure differential between the chambers, which also holdsa cell in place once the cell has been drawn to the opening. Aconvenient method of imposing such a pressure differential is tomaintain atmospheric pressure in the upper chamber while lowering thepressure in the lower chamber below atmospheric by attaching a syringeto the lower chamber and pulling on the syringe plunger. Care should betaken to limit the pressure differential to one that will not damage thecell.

FIGS. 3 a and 3 b illustrate to a different apparatus and method withinthe scope of this invention. This apparatus and method involve a fluidsuspension of biological cells flowing through a conduit or flowchannel, in which the cells pass through a region between a pair ofelectrodes. The longitudinal cross section of FIG. 3 a shows the walls41 of the channel, and a biological cell 42 passing downward through thelumen of the channel (in the direction of the arrow). The transversecross section of FIG. 3 b shows that the channel is rectangular in crosssection, although other cross-sectional geometries may be used.Electrodes 43, 44 are formed as coatings on two opposing walls of thechannel. The electrodes are connected through leads to a printed circuitboard 45 which measures the impedance and controls the voltage appliedto the electrodes. The biological cell 42 is shown passing through theregion between the two electrodes.

The area of the cross section of the channel is large enough to permitthe cell to pass through essentially unimpeded by the channel walls, andyet small enough that only one cell can pass through the inter-electroderegion at a time. In addition, each electrode 43, 44 is eitherapproximately equal in length or slightly larger in length than thediameter of the biological cell, so that the cell upon entering theregion causes a significant or measurable decrease in the currentpassing through the region due to the voltage applied across electrodes.The spacing of the electrodes, i.e., the distance between them, islikewise subject to the same considerations. The biological cells aresuspended in a liquid solution of the species to be introduced into thecells, and the suspension is passed through the channel. A voltage isapplied between the electrodes as suspension flows through the channel,and the current between the electrodes (or the impedance) is monitored.A significant drop in the current indicates the presence of a biologicalcell in the inter-electrode region. Once the cell is detected in thismanner, an electroporation pulse can be applied to the electrodes whilethe cell is still in the inter-electrode region, and impedance can beobserved further to detect the onset of electroporation. The speciesdissolved in the liquid solution will enter the cell as a result of theelectroporation.

Variations on these structures and methods will be readily apparent tothose skilled in the art. For example, the barriers described above canbe minimized or avoided by using microelectrodes that are the same sizeas or smaller than the biological cells. Examples of suchmicroelectrodes are carbon fiber microelectrodes (such as ProCFE, AxonInstruments, Foster City, Calif., USA) used in conjunction withhigh-graduation micromanipulators (such as those available fromNarishige MWH-3, Tokyo, Japan). Microelectrodes can be used in place ofthe electrodes shown in FIG. 2 or in place of those shown in FIGS. 3 aand 3 b.

The following examples are offered for illustration, and are notintended to impose limits on the scope of the invention.

EXAMPLE 1

A series of experiments was performed using a microelectroporationsystem consisting of the microelectroporation device described above andshown in FIG. 2, combined with flow and pressure control units andpressure gauges for the liquids to be circulated through the upper andlower chambers, a variable DC power supply, a pulse generator and poweramplifier for imposing voltage pulses across the device, a digitaloscilloscope for monitoring the pulses, a fluorescent microscope, a CCD(charge coupled device) camera, and a computer with image processing andwaveform processing software. Both chambers of the device were filledwith physiological saline and cells were introduced into the upperchamber. Liquid motion in the top and bottom chambers was controlled bysyringes. The pressure in the upper chamber was atmospheric while thepressure in the lower chamber was reduced below atmospheric by pullingon the barrel of the syringe connected to that chamber. The voltage wasapplied in single square pulses ranging from zero to 120V in magnitudeand from 2 microseconds to 100 milliseconds in duration. The distancebetween the electrodes in the upper and lower chambers was 900 microns.

The tests in this example were performed using ND-1 human prostateadenocarcinoma cells with a typical diameter of 20 microns. The openingin the microelectroporation device was 5 microns in diameter. Arectangular voltage pulse was applied with a duration of 60milliseconds, and the pulse was applied at various amplitudes rangingfrom 10V to 60V in increments of 5 volts. With each pulse, the electriccurrent passing through the opening was measured. Experiments wereperformed with the cells and were repeated both with the opening stoppedby a glass bead and with no obstruction at all in the opening. Theresults in each case were expressed as microamperes of current vs. voltsof pulse amplitude and are plotted in FIG. 4, in which the upper curve(data points represented by x's) represents the unobstructed opening,the lower curve (data points represented by asterisks) represents thedata taken with the glass bead residing in the opening, and the threemiddle curves (open squares, open upright triangles, and open invertedtriangles) represent data taken with three different ND-1 cells residingin the opening.

The upper curve shows that the current increases in a substantiallysteady manner as the voltage increases when there is no barrier to thepassage of current through the opening. The lower curve also shows asubstantially steady rise as the voltage increases, although at a muchlower level. The current values shown in the lower curve represent straycurrents through the device. The curves of data taken with the ND-1cells across the opening show that at low voltages the current is closein value to that obtained when the opening is closed by the glass beadwhile at high voltages the current rises to the levels obtained with anunobstructed opening. The transition is a sharp increase which isindicative of the formation of pores in the cell membrane through whichan electric current can pass, i.e., the onset of electroporation. In allthree cells, the transition occurred at voltages between 30V and 40V. Intwo of the three cells (open squares and open upright triangles), theonset of electroporation occurred essentially at the same voltage, whilein the third (inverted triangles), the onset occurred at a voltage thatwas lower than the other two by about 5V. This illustrates the value ofcontrolling the process for individual cells to achieve optimal results.

After the data shown in FIG. 4 was generated, the pulses were reappliedin descending order of amplitude values, and the resulting curvesdisplayed hysteresis, i.e., the curves obtained with descendingamplitudes were higher in voltage than those obtained with ascendingamplitudes. This indicated that the electroporation in these experimentswas irreversible.

EXAMPLE 2

Using the same microelectroporation system used in Example 1, a seriesof tests were performed on rat hepatocytes (ATCC #CRL-1439), whosetypical cell diameter was 20 microns, the microelectroporation apparatushaving an opening that was 4 microns in diameter. Here as well,rectangular voltage pulses that were 60 milliseconds in duration wereused, ranging in amplitude from 10V to 37.5V in increments of 5V in theportion from 10V to 30V and in increments of 2.5V in the portion from30V to 37.5V. The experiments were performed in some cases only byincreasing the amplitudes and in others by first increasing, thendecreasing the amplitudes to evaluate reversibility. The results areplotted in the graphs shown in FIGS. 5 a, 5 b, 5 c, and 5 d. In eachcase, the upper curve (data points represented by circles) is the datataken with neither a cell nor a glass bead residing in the opening, thelower curve (data points represented by squares) is the data taken witha glass bead in the opening, and the middle curve (data pointsrepresented by triangles) is the data taken with a hepatocyte in theopening, using different hepatocytes for each of the four Figures.

In FIG. 5 a, the amplitude was increased and not decreased, displayingan electroporation threshold voltage of between 25V and 30V. In FIGS. 5b and 5 c, the amplitude was first increased and then decreased toproduce the two middle curves. Although the ascending and descendingcurves are not differentiated, they are substantially identical in eachFigure, indicating that the cell membrane in each of these two casesresealed after each voltage pulse and thus that the pore formation wasreversible. In the test represented by FIG. 5 d, the cell disintegratedonce the applied voltage-exceeded 37.5V, although this is not shown inthe Figure. It is significant to note that despite the fact that thesame cell types were used in each of FIGS. 5 a, 5 b, 5 c, and 5 d, theelectroporation threshold voltage differed among the individual cells,although all were within the range of 20V to 35V. Adaptation of theprocedure to individual cells is readily achieved by monitoring thecurrent in this manner to note when the electroporation thresholdoccurs. Selection of the optimal exposure time, voltage, compositionchanges in the surrounding liquids, and other parameters of the systemcan then be made to achieve the desired treatment of the cell withoutdestruction of the cell.

The methods described herein are useful tools in the laboratory forconducting fundamental research in the electroporation properties ofbiological cells, and useful tools in industry for processing largequantities of cells in a flow-through manner. By enabling one to observeand record the current flowing through individual cells, one can controlthe amplitude and duration of the voltage pulse to achieve optimalresults. In addition, the devices described and shown herein for use inpracticing the invention can be constructed with transparent parts andof a size suitable for mounting on a microscope stage. This will permitone to correlate the electrical current measurements to visualobservations and fluorescence measurements inside the cell. The devicecan be used to electrically detect, through the measurement of currents,the point in time when a cell becomes lodged in the opening as well asthe point in time when pore formation is achieved in the cell membrane.For larger scale and industrial applications, large numbers ofmicroelectroporation devices of the type described herein can bearranged in parallel. For each cell, electrical information indicatingthe trapping of a cell in the opening (such as a sharp drop in thecurrent) can be used to generate a signal that will initiate anelectroporation sequence, and further electrical information indicatingthe completion of electroporation (such as a sharp rise in current) willgenerate a signal that will release the cell (for example by eliminatingor reversing the pressure differential) and permit the next cell to flowtoward the opening.

Further implementations, applications, adaptations, and embodiments ofthe concepts, features and methods described herein that are within thescope of this invention will be readily apparent to those skilled in theart.

1. A method of electroporating a cell mass in a controlled manner, themethod comprising: (a) applying a voltage across a cell mass; (b)detecting the ratio of electric current through the cell mass to thevoltage applied across the cell mass, wherein a change in the ratioindicates a change in the degree of electroporation of the cell mass;(c) determining an irreversible electroporation voltage for the cells ofthe cell mass, wherein the determining of the irreversibleelectroporation voltage comprises determining an electroporationcurrent-to-voltage onset point of the cell mass or an electroporationcurrent-to-voltage hysteresis of the cell mass; and (d) adjusting theapplied voltage to provide the determined irreversible electroporationvoltage of step (c), thereby providing an irreversible electroporationof the cells of the cell mass.
 2. The method of claim 1, wherein in step(c) the determining of the irreversible electroporation voltage furthercomprises continuously detecting a current-to-voltage ratio in the cellmass, and wherein in step (d) the adjusting further comprises adjustingthe applied voltage for a duration of time sufficient to provide acontrolled, irreversible electroporation of cells of the cell mass. 3.The method of claim 1, wherein the cells are human prostate cancercells.
 4. The method of claim 1, further comprising: (e) positioning abarrier having an opening wherein the positioning of the barriercomprises trapping the cell mass in the opening of the barrier, therebycausing the electrical current to flow through the trapped cell mass. 5.A method of electroporating a biological cell sample in a controlledmanner, the method comprising: (a) applying a voltage across abiological cell sample; (b) detecting changes in electrical impedanceoccurring across the biological cell sample, wherein said changes inelectrical impedance are caused by the applied voltage electroporatingcells in the biological cell sample; (c) calculating from the changes inelectrical impedance obtained from step (b) an amount and a duration ofa voltage to obtain a change in electrical impedance; and (d) from theamount and duration of voltage calculated in step (c), applying anamount and a duration of a voltage sufficient to obtain a controlled,irreversible electroporation of the cells of the biological cell sample.6. The method of claim 5, wherein the biological cell sample is a tissuesample containing the biological cells and wherein the biological cellsin the sample are human prostate adenocarcinoma cells.
 7. The method ofclaim 5, further comprising: (e) positioning a barrier having an openingwherein the positioning of the barrier comprises trapping the cellsample in the opening of the barrier and wherein the applying thevoltage causes the electrical current to flow through the trapped cellsample.